Overexpression of normal and mutant Arp1alpha (centractin) differentially affects microtubule organization during mitosis and interphase

1999 ◽  
Vol 112 (20) ◽  
pp. 3507-3518 ◽  
Author(s):  
I.B. Clark ◽  
D.I. Meyer
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Cytoplasmic Dynein ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
Mitotic Apparatus ◽  
Mutant Proteins ◽  
Cell Extracts ◽  
Dynactin Complex

Dynactin is a large multisubunit complex that regulates cytoplasmic dynein-mediated functions. To gain insight into the role of dynactin's most abundant component, Arp1alpha was transiently overexpressed in mammalian cells. Arp1alpha overexpression resulted in a cell cycle delay at prometaphase. Intracellular dynactin, dynein and nuclear/mitotic apparatus (NuMA) protein were recruited to multiple foci associated with ectopic cytoplasmic aggregates of Arp1alpha in transfected cells. These ectopic aggregates nucleated supernumerary microtubule asters at prometaphase. Point mutations were generated in Arp1alpha that identified specific amino acids required for the prometaphase delay and for the formation of supernumerary microtubule asters. The mutant Arp1alpha proteins formed aggregates in cells that colocalized with dynactin and dynein peptides, but in contrast to wild-type Arp1alpha, NuMA localization remained unaffected. Although expression of mutant Arp1alpha proteins had no effect on mitotic cells, in interphase cells expression of the mutants resulted in disruption of the microtubule network. Immunoprecipitation studies demonstrated that overexpressed Arp1alpha interacts with dynactin and NuMA proteins in cell extracts, and that these interactions are destabilized in the Arp1alpha mutants. We conclude that the amino acids altered in the Arp1alpha mutant proteins participate in stabilizing interactions between overexpressed Arp1alpha and components of the endogenous dynactin complex as well as the NuMA protein.

scholarly journals Dynactin Is Required for Microtubule Anchoring at Centrosomes

1999 ◽  
Vol 147 (2) ◽  
pp. 321-334 ◽  
Author(s):  
N.J. Quintyne ◽  
S.R. Gill ◽  
D.M. Eckley ◽  
C.L. Crego ◽  
D.A. Compton ◽  
...  
Keyword(s):  
Mitotic Cell ◽  
Cytoplasmic Dynein ◽  
Microtubule Organization ◽  
Cultured Fibroblasts ◽  
Cell Extracts ◽  
Dynein Motor ◽  
Mitotic Spindle Assembly ◽  
Dynactin Complex

The multiprotein complex, dynactin, is an integral part of the cytoplasmic dynein motor and is required for dynein-based motility in vitro and in vivo. In living cells, perturbation of the dynein–dynactin interaction profoundly blocks mitotic spindle assembly, and inhibition or depletion of dynein or dynactin from meiotic or mitotic cell extracts prevents microtubules from focusing into spindles. In interphase cells, perturbation of the dynein–dynactin complex is correlated with an inhibition of ER-to-Golgi movement and reorganization of the Golgi apparatus and the endosome–lysosome system, but the effects on microtubule organization have not previously been defined. To explore this question, we overexpressed a variety of dynactin subunits in cultured fibroblasts. Subunits implicated in dynein binding have effects on both microtubule organization and centrosome integrity. Microtubules are reorganized into unfocused arrays. The pericentriolar components, γ tubulin and dynactin, are lost from centrosomes, but pericentrin localization persists. Microtubule nucleation from centrosomes proceeds relatively normally, but microtubules become disorganized soon thereafter. Overexpression of some, but not all, dynactin subunits also affects endomembrane localization. These data indicate that dynein and dynactin play important roles in microtubule organization at centrosomes in fibroblastic cells and provide new insights into dynactin–cargo interactions.

scholarly journals Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued.

1995 ◽  
Vol 131 (6) ◽  
pp. 1507-1516 ◽  
Author(s):  
K T Vaughan ◽  
R B Vallee
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Direct Interaction ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Cell Extracts ◽  
Complex Protein ◽  
Microtubule Motor ◽  
Dynactin Complex ◽  
Intermediate Chains

Cytoplasmic dynein is a retrograde microtubule motor thought to participate in organelle transport and some aspects of minus end-directed chromosome movement. The mechanism of binding to organelles and kinetochores is unknown. Based on homology with the Chlamydomonas flagellar outer arm dynein intermediate chains (ICs), we proposed a role for the cytoplasmic dynein ICs in linking the motor protein to organelles and kinetochores. In this study two different IC isoforms were used in blot overlay and immunoprecipitation assays to identify IC-binding partners. In overlays of complex protein samples, the ICs bound specifically to polypeptides of 150 and 135 kD, identified as the p150Glued doublet of the dynactin complex. In reciprocal overlay assays, p150Glued specifically recognized the ICs. Immunoprecipitations from total Rat2 cell extracts, rat brain cytosol, and rat brain membranes further identified the dynactin complex as a specific target for IC binding. using truncation mutants, the sites of interaction were mapped to amino acids 1-123 of IC-1A and amino acids 200-811 of p150Glued. While cytoplasmic dynein and dynactin have been implicated in a common pathway by genetic analysis, our findings identify a direct interaction between two specific component polypeptides and support a role for dynactin as a dynein "receptor". Our data also suggest, however, that this interaction must be highly regulated.

scholarly journals Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells

2002 ◽  
Vol 156 (1) ◽  
pp. 87-100 ◽  
Author(s):  
Toshiro Ohta ◽  
Russell Essner ◽  
Jung-Hwa Ryu ◽  
Robert E. Palazzo ◽  
Yumi Uetake ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Amino Acid Residues ◽  
Protein Overexpression ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
Centrosomal Protein ◽  
Wide Range ◽  
Spindle Microtubules

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.

scholarly journals Evidence for dynein and astral microtubule–mediated cortical release and transport of Gαi/LGN/NuMA complex in mitotic cells

2013 ◽  
Vol 24 (7) ◽  
pp. 901-913 ◽  
Author(s):  
Zhen Zheng ◽  
Qingwen Wan ◽  
Jing Liu ◽  
Huabin Zhu ◽  
Xiaogang Chu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescence Recovery After Photobleaching ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Spindle Positioning ◽  
Spindle Poles ◽  
Astral Microtubule ◽  
Mammalian Homologue ◽  
Cortical Localization

Spindle positioning is believed to be governed by the interaction between astral microtubules and the cell cortex and involve cortically anchored motor protein dynein. How dynein is recruited to and regulated at the cell cortex to generate forces on astral microtubules is not clear. Here we show that mammalian homologue of Drosophila Pins (Partner of Inscuteable) (LGN), a Gαi-binding protein that is critical for spindle positioning in different systems, associates with cytoplasmic dynein heavy chain (DYNC1H1) in a Gαi-regulated manner. LGN is required for the mitotic cortical localization of DYNC1H1, which, in turn, also modulates the cortical accumulation of LGN. Using fluorescence recovery after photobleaching analysis, we show that cortical LGN is dynamic and the turnover of LGN relies, at least partially, on astral microtubules and DYNC1H1. We provide evidence for dynein- and astral microtubule–mediated transport of Gαi/LGN/nuclear mitotic apparatus (NuMA) complex from cell cortex to spindle poles and show that actin filaments counteract such transport by maintaining Gαi/LGN/NuMA and dynein at the cell cortex. Our results indicate that astral microtubules are required for establishing bipolar, symmetrical cortical LGN distribution during metaphase. We propose that regulated cortical release and transport of LGN complex along astral microtubules may contribute to spindle positioning in mammalian cells.

scholarly journals Specificity of Cytoplasmic Dynein Subunits in Discrete Membrane-trafficking Steps

2009 ◽  
Vol 20 (12) ◽  
pp. 2885-2899 ◽  
Author(s):  
Krysten J. Palmer ◽  
Helen Hughes ◽  
David J. Stephens
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Cytoplasmic Dynein ◽  
Dominant Negative ◽  
Automated Image Analysis ◽  
Recycling Endosome ◽  
Microtubule Network ◽  
Dynein Motor ◽  
Specific Subset ◽  
Biochemical Analyses

The cytoplasmic dynein motor complex is known to exist in multiple forms, but few specific functions have been assigned to individual subunits. A key limitation in the analysis of dynein in intact mammalian cells has been the reliance on gross perturbation of dynein function, e.g., inhibitory antibodies, depolymerization of the entire microtubule network, or the use of expression of dominant negative proteins that inhibit dynein indirectly. Here, we have used RNAi and automated image analysis to define roles for dynein subunits in distinct membrane-trafficking processes. Depletion of a specific subset of dynein subunits, notably LIC1 (DYNC1LI1) but not LIC2 (DYNC1LI2), recapitulates a direct block of ER export, revealing that dynein is required to maintain the steady-state composition of the Golgi, through ongoing ER-to-Golgi transport. Suppression of LIC2 but not of LIC1 results in a defect in recycling endosome distribution and cytokinesis. Biochemical analyses also define the role of each subunit in stabilization of the dynein complex; notably, suppression of DHC1 or IC2 results in concomitant loss of Tctex1. Our data demonstrate that LIC1 and LIC2 define distinct dynein complexes that function at the Golgi versus recycling endosomes, respectively, suggesting that functional populations of dynein mediate discrete intracellular trafficking pathways.

scholarly journals The Role of HCV E2 Protein Glycosylation in Functioning of Virus Envelope Proteins in Insect and Mammalian Cells

Acta Naturae ◽  
2015 ◽  
Vol 7 (1) ◽  
pp. 87-97 ◽  
Author(s):  
O. V. Orlova ◽  
V. L. Drutsa ◽  
P. V. Spirin ◽  
A. V. Ivanov ◽  
V. S. Prasolov ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Expression System ◽  
Point Mutations ◽  
Envelope Proteins ◽  
Protein Glycosylation ◽  
Virus Envelope ◽  
Mutant Proteins ◽  
Virus Like Particles ◽  
E2 Glycoprotein ◽  
Glycosylation Sites

The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.

RNA-recognition motifs and glycine and arginine-rich region cooperatively regulate the nucleolar localization of nucleolin

2020 ◽  
Author(s):  
Mitsuru Okuwaki ◽  
Ai Saotome-Nakamura ◽  
Masashi Yoshimura ◽  
Shoko Saito ◽  
Hiroko Hirawake-Mogi ◽  
...  
Keyword(s):  
Rna Binding ◽  
Point Mutations ◽  
Binding Activity ◽  
Nucleolar Localization ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Mutant Proteins ◽  
Cell Extracts ◽  
Nucleolar Proteins ◽  
Recognition Motifs

Abstract Nucleolin (NCL) is a nucleolar protein that is involved in the regulation of the nucleolar structure and functions, and consists of three distinct regions: the N-terminal region; the middle region, which contains four RNA-recognition motifs (RRMs); and the C-terminal glycine and arginine-rich (GAR) region. The primary function of the RRMs and GAR is thought to be specific RNA binding. However, it is not well understood how these RNA-binding regions of NCL separately or cooperatively regulate its nucleolar localization and functions. To address this issue, we constructed mutant proteins carrying point mutations at the four RRMs individually or deletion of the C-terminal GAR region. We found that the GAR deletion and the mutations in the fourth RRM (RRM4) decreased the nucleolar localization of NCL. Biochemical analyses showed that NCL interacted directly with ribosomal RNAs (rRNAs) and G-rich oligonucleotides, and that this interaction was decreased by mutations at RRM1 and RRM4 and GAR deletion. Although GAR deletion decreased the rRNA-binding activity of NCL, the mutant was efficiently co-precipitated with rRNAs and nucleolar proteins from cell extracts. These contradictory results suggest that NCL stably localizes to the nucleoli via the interactions with rRNAs and nucleolar proteins via GAR, RRM1, and RRM4.

scholarly journals Cytoplasmic Dynein–Dynactin Complex Is Required for Spermatid Growth but Not Axoneme Assembly in Drosophila

2004 ◽  
Vol 15 (5) ◽  
pp. 2470-2483 ◽  
Author(s):  
Anindya Ghosh-Roy ◽  
Madhura Kulkarni ◽  
Vikash Kumar ◽  
Seema Shirolikar ◽  
Krishanu Ray
Keyword(s):  
Cell Growth ◽  
Light Chain ◽  
Cytoplasmic Dynein ◽  
Partial Reduction ◽  
Dynein Light Chain ◽  
Microtubule Organization ◽  
Radial Spoke ◽  
Dynein Arms ◽  
Spectrin Cytoskeleton ◽  
Dynactin Complex

Spermatids derived from a single gonial cell remain interconnected within a cyst and elongate by synchronized growth inside the testis in Drosophila. Cylindrical spectrin-rich elongation cones form at their distal ends during the growth. The mechanism underlying this process is poorly understood. We found that developing sperm tails were abnormally coiled at the growing ends inside the cysts in the Drosophila Dynein light chain 1 (ddlc1) hemizygous mutant testis. A quantitative assay showed that average number of elongation cones was reduced, they were increasingly deformed, and average cyst lengths were shortened in ddlc1 hemizygous testes. These phenotypes were further enhanced by additional partial reduction of Dhc64C and Glued and rescued by Myc-PIN/LC8 expression in the gonial cells in ddlc1 backgrounds. Furthermore, DDLC1, DHC, and GLUED were enriched at the distal ends of growing spermatids. Finally, ultrastructure analysis of ddlc1 testes revealed abnormally formed interspermatid membrane, but the 9 + 2 microtubule organization, the radial spoke structures, and the Dynein arms of the axoneme were normal. Together, these findings suggest that axoneme assembly and spermatid growth involve independent mechanisms in Drosophila and DDLC1 interacts with the Dynein–Dynactin complex at the distal ends of spermatids to maintain the spectrin cytoskeleton assembly and cell growth.

scholarly journals Point mutations that inactivate MGAT4D-L, an inhibitor of MGAT1 and complex N-glycan synthesis

2020 ◽  
Vol 295 (41) ◽  
pp. 14053-14064
Author(s):  
Ayodele Akintayo ◽  
Joshua Mayoral ◽  
Masahiro Asada ◽  
Jian Tang ◽  
Subha Sundaram ◽  
...  
Keyword(s):  
Amino Acids ◽  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Mechanism Of Inhibition ◽  
C Terminus ◽  
Membrane Bound ◽  
Galanthus Nivalis ◽  
Glcnac Transferase

The membrane-bound, long form of MGAT4D, termed MGAT4D-L, inhibits MGAT1 activity in transfected cells and reduces the generation of complex N-glycans. MGAT1 is the GlcNAc-transferase that initiates complex and hybrid N-glycan synthesis. We show here that Drosophila MGAT1 was also inhibited by MGAT4D-L in S2 cells. In mammalian cells, expression of MGAT4D-L causes the substrate of MGAT1 (Man5GlcNAc2Asn) to accumulate on glycoproteins, a change that is detected by the lectin Galanthus nivalis agglutinin (GNA). Using GNA binding as an assay for the inhibition of MGAT1 in MGAT4D-L transfectants, we performed site-directed mutagenesis to determine requirements for MGAT1 inhibition. Deletion of 25 amino acids (aa) from the C terminus inactivated MGAT4D-L, but deletion of 20 aa did not. Conversion of the five key amino acids (PSLFQ) to Ala, or deletion of PSLFQ in the context of full-length MGAT4D-L, also inactivated MGAT1 inhibitory activity. Nevertheless, mutant, inactive MGAT4D-L interacted with MGAT1 in co-immuno-precipitation experiments. The PSLFQ sequence also occurs in MGAT4A and MGAT4B GlcNAc-transferases. However, neither inhibited MGAT1 in transfected CHO cells. MGAT4D-L inhibitory activity could be partially transferred by attaching PSLFQ or the 25-aa C terminus of MGAT4D-L to the C terminus of MGAT1. Mutation of each amino acid in PSLFQ to Ala identified both Leu and Phe as independently essential for MGAT4D-L activity. Thus, replacement of either Leu-395 or Phe-396 with Ala led to inactivation of MGAT4D-L inhibitory activity. These findings provide new insights into the mechanism of inhibition of MGAT1 by MGAT4D-L, and for the development of small molecule inhibitors of MGAT1.

scholarly journals Point Mutations in the WD40 Domain of Eed Block Its Interaction with Ezh2

1998 ◽  
Vol 18 (10) ◽  
pp. 5634-5642 ◽  
Author(s):  
Oleg Denisenko ◽  
Maria Shnyreva ◽  
Hideaki Suzuki ◽  
Karol Bomsztyk
Keyword(s):  
Mammalian Cells ◽  
Protein A ◽  
Point Mutations ◽  
Full Length ◽  
Cdna Clones ◽  
Mouse Development ◽  
Cell Extracts ◽  
Terminal Fragment ◽  
Two Hybrid

ABSTRACT The Polycomb group proteins are involved in maintenance of the silenced state of several developmentally regulated genes. These proteins form large aggregates with different subunit compositions. To explore the nature of these complexes and their function, we used the full-length Eed (embryonic ectoderm development) protein, a mammalian homolog of the Drosophila Polycomb group protein Esc, as a bait in the yeast two-hybrid screen. Several strongly interacting cDNA clones were isolated. The cloned cDNAs all encoded the 150- to 200-amino-acid N-terminal fragment of the mammalian homolog of the Drosophila Enhancer of zeste [E(z)] protein, Ezh2. The full-length Ezh2 bound strongly to Eed in vitro, and Eed coimmunoprecipitated with Ezh2 from murine 70Z/3 cell extracts, confirming the interaction between these proteins observed in yeast. Mutations T1031A and T1040C in one of the WD40 repeats of Eed, which account for the hypomorphic and lethal phenotype of eed in mouse development, blocked binding of Ezh2 to Eed in a two-hybrid interaction in yeast and in mammalian cells. These mutations also blocked the interaction between these proteins in vitro. In mammalian cells, the Gal4-Eed fusion protein represses the activity of a promoter bearing Gal4 DNA elements. The N-terminal fragment of the Ezh2 protein abolished the transcriptional repressor activity of Gal4-Eed protein when they were coexpressed in mammalian cells. Eed and Ezh2 were also found to bind RNA in vitro, and RNA altered the interaction between these proteins. These findings suggest that Polycomb group proteins Eed and Ezh2 functionally interact in mammalian cells, an interaction that is mediated by the WD40-containing domain of Eed protein.

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